Screening Of Elastase Inhibitory Peptide From Walnut Meal And Preparation Of Peptide-loaded Nanoparticles

Walnut meal is the by-product of the walnut oil industry,which has high protein content and varieties of amino acids.It can improve the economic benefit by preparing functional bioactive peptides from the walnut meal.Elastase is a protein hydrolase in the human body,which causes skin aging,lung disease,and rheumatoid arthritis through over-degradation of elastin.Thus,inhibition of elastase could promote health by preventing the excessive hydrolysis of elastin.Previous studies showed that peptide from walnut has the potential of anti-skin photoaging,but its effect on elastase is not known.Also,there are some challenges in peptide applications.Therefore,it is helpful to explore the colloidal delivery system of peptides to improve the feasibility of its commercialization.In this study,the walnut meal was used to prepare bioactive peptides with elastase inhibitory activity by ultrasonic enzymatic hydrolysis.Alkaline protease was selected as hydrolase.Besides,the single factor experiments and response surface methodology were used to optimize the conditions of peptide extraction.The optimum conditions were as follows:liquid to material ratio was 27:1（v/w）,ultrasonic power was 410 W,Alkaline protease amount was 3.3%（w/w）,time was 4.3 h.Under such a setting,the yield of the peptide was 52.75±1.64%,and the IC₅₀ values of enzymatic hydrolysate is2.680±0.127 mg/m L.The polypeptides of the walnut meal were purified by ultrafiltration.The 0～3 k Da component with the highest activity was analyzed by LC-MS/MS and 556 peptide sequences were obtained.Peptide FFVPF was screened by Py Rx virtual screening and Autodock Vina molecular docking.The computer simulation result showed there are hydrophobic interaction,hydrogen bond and pi-sulfur interaction between FFVPF and elastase.Furthermore,peptide NSLNLPIL was screened by amino acid composition analysis result and literature reviews.FFVPF and NSLNLPIL both have strong elastase inhibition ability.The IC₅₀ values of FFVPF and NSLNLPIL were 0.469±0.010 mg/m L and0.635±0.030 mg/m L,respectively.The retention rate of FFVPF was 87.48±1.24%after simulated gastric digestion and it was completely decomposed after digestion in simulated intestinal.The peptide-loaded CS-TPP nanoparticles were prepared by the ionotropic gelation method and characterized.The maximum encapsulation efficiency could reach 94.58±0.23%,and resultant particle sizes ranged from 140 to 180 nm,PDI values from 0.2 to 0.25,and Zeta potential values from+33m V to+36 m V.The morphology of FFVPF-loaded nanoparticles showed spherical shapes with a slightly pointed surface.The formation of CS-TPP nanoparticles was based on hydrogen bonding and electrostatic interaction,and the peptides interacted with CS-TPP NPs through hydrogen bonds.CS-TPP NP has a crystalline structure,and the crystal form of NPs were not changed after entrapping peptides.Besides,CS-TPP NPs have good stability in different p H,there is no leakage of the peptide in the range of p H 4～6.The retention rate could reach more than 70%from p H 2 to 8,which demonstrates CS-TPP system could improve the gastrointestinal stability of the peptide.CS-TPP NPs could maintain stability when the temperature is no more than 65℃,but it is sensitive to UV-light.In addition,FFVPF-loaded NPs showed a rapid initial burst in the in vitro drug release test,and then the release speed decreased with time.In this study,2 peptides with high elastase inhibitory activity were identified and screened from walnut meal polypeptide,and the colloidal delivery system loaded bioactive peptides was established.This research is helpful to realize the application of peptides and other hydrophilic functional factors in food industry.